DNA-induced alpha-helix capping in conserved linker sequences is a determinant of binding affinity in Cys(2)-His(2) zinc fingers

High-affinity, sequence-specific DNA binding by Cys(2)-His(2) zinc finger proteins is mediated by both specific protein-base interactions and non-specific contacts between charged side-chains and the phosphate backbone. In addition, in DNA complexes of multiple zinc fingers, protein-protein interactions between the finger units contribute to the binding affinity. We present NMR evidence for another contribution to high- affinity binding, a highly specific DNA-induced helix capping involving residues in the linker sequence between fingers. Capping at the C terminus of the alpha-helix in each zinc finger, incorporating a consensus TGEKP linker sequence that follows each finger, provides substantial binding energy to the DNA complexes of zinc fingers 1-3 of TFIIIA (zf1-3) and the four zinc fingers of the Wilms' tumor suppressor protein (wt1-4). The same alpha-helix C-capping motif is observed in the X-ray structures of four other protein-DNA complexes. The structures of each of the TGEKP linkers in these complexes can be superimposed on the linker sequences in the zf1-3 complex, revealing a remarkable similarity in both backbone and side-chain conformations. The canonical linker structures from the zinc-finger-DNA complexes have been compared to the NMR structure of the TGEKP linker connecting fingers 1 and 2 in zf1-3 in the absence of DNA. This comparison reveals that additional stabilization likely arises in the DNA complexes from hydrogen bonding between the backbone amide of E3 and the side-chain O(gamma) of T1 in the linker. We suggest that these DNA-induced C-capping interactions provide a means whereby the multiple-finger complex, which must necessarily be domain-flexible in the unbound state as it searches for the correct DNA sequence, can be "snap-locked" in place once the correct DNA sequence is encountered. These observations provide a rationale for the high conservation of the TGEKP linker sequences in Cys(2)-His(2) zinc finger proteins.     

Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA

The zinc finger domain of the Wilms tumor suppressor protein (WT1) contains four canonical Cys(2)His(2) zinc fingers. WT1 binds preferentially to DNA sequences that are closely related to the EGR-1 consensus site. We report the structure determination by both X-ray crystallography and NMR spectroscopy of the WT1 zinc finger domain in complex with DNA. The X-ray structure was determined for the complex with a cognate 14 base-pair oligonucleotide, and composite X-ray/NMR structures were determined for complexes with both the 14 base-pair and an extended 17 base-pair DNA. This combined approach allowed unambiguous determination of the position of the first zinc finger, which is influenced by lattice contacts in the crystal structure. The crystal structure shows the second, third and fourth zinc finger domains inserted deep into the major groove of the DNA where they make base-specific interactions. The DNA duplex is distorted in the vicinity of the first zinc finger, with a cytidine twisted and tilted out of the base stack to pack against finger 1 and the tip of finger 2. By contrast, the composite X-ray/NMR structures show that finger 1 continues to follow the major groove in the solution complexes. However, the orientation of the helix is non-canonical, and the fingertip and the N terminus of the helix project out of the major groove; as a consequence, the zinc finger side-chains that are commonly involved in base recognition make no contact with the DNA. We conclude that finger 1 helps to anchor WT1 to the DNA by amplifying the binding affinity although it does not contribute significantly to binding specificity. The structures provide molecular level insights into the potential consequences of mutations in zinc fingers 2 and 3 that are associated with Denys-Drash syndrome and nephritic syndrome. The mutations are of two types, and either destabilize the zinc finger structure or replace key base contact residues.     

Combining structure-based design with phage display to create new Cys(2)His(2) zinc finger dimers

BACKGROUND: Several strategies have been reported for the design and selection of novel DNA-binding proteins. Most of these studies have used Cys(2)His(2) zinc finger proteins as a framework, and have focused on constructs that bind DNA in a manner similar to Zif268, with neighboring fingers connected by a canonical (Krüppel-type) linker. This linker does not seem ideal for larger constructs because only modest improvements in affinity are observed when more than three fingers are connected in this manner. Two strategies have been described that allow the productive assembly of more than three canonically linked fingers on a DNA site: connecting sets of fingers using linkers (covalent), or assembling sets of fingers using dimerization domains (non-covalent). RESULTS: Using a combination of structure-based design and phage display, we have developed a new dimerization system for Cys(2)His(2) zinc fingers that allows the assembly of more than three fingers on a desired target site. Zinc finger constructs employing this new dimerization system have high affinity and good specificity for their target sites both in vitro and in vivo. Constructs that recognize an asymmetric binding site as heterodimers can be obtained through substitutions in the zinc finger and dimerization regions. CONCLUSIONS: Our modular zinc finger dimerization system allows more than three Cys(2)His(2) zinc fingers to be productively assembled on a DNA-binding site. Dimerization may offer certain advantages over covalent linkage for the recognition of large DNA sequences. Our results also illustrate the power of combining structure-based design with phage display in a strategy that assimilates the best features of each method.     

Effects of bulkiness and hydrophobicity of an aliphatic amino acid in the recognition helix of the GAGA zinc finger on the stability of the hydrophobic core and DNA binding affinity

The GAGA factor of Drosophila melanogaster uses a single Cys 2His 2-type zinc finger for specific DNA binding. The conformation and DNA binding mode of the GAGA zinc finger are similar to those of other structurally characterized zinc fingers. In almost all Cys 2His 2-type zinc fingers, the fourth position of the DNA-recognizing helix is occupied by the Leu residue involved in the formation of the minimal hydrophobic core. However, no systematic study on the precise role of the Leu residue in the hydrophobic core formation and DNA binding function has been reported. In this study, the Leu residue is substituted with other aliphatic amino acids having different side chain lengths and hydrophobicities, namely, Ile, Val, Aib, and Ala. The metal binding properties were studied by UV-vis spectroscopy. The peptide conformations were examined by CD and NMR spectroscopies. Furthermore, the DNA binding ability was examined with a gel mobility shift assay. Though the Ile, Val, and Aib mutants exhibited conformations similar to those of the wild type, the DNA binding affinity decreased as the side chain length of the amino acid decreased. Interestingly, the Val mutant can bind to the cognate DNA, while Aib cannot, in spite of the similarity in their secondary structures based on the CD measurements. Variable-temperature NMR experiments clearly indicated differences in the stability of the hydrophobic core between the Val and Aib mutants. This study demonstrates that the bulkiness of the conserved aliphatic residue is important in the formation of the well-packed minimal hydrophobic core and proper ternary structure and that the hydrophobic core stabilization is apparently related to the DNA binding function of the GAGA zinc finger.     

Contributions of cysteine residues in Zn2 to zinc fingers and thiol-disulfide oxidoreductase activities of chaperone DnaJ

Escherichia coli DnaJ, possessing both chaperone and thiol-disulfide oxidoreductase activities, is a homodimeric Hsp40 protein. Each subunit contains four copies of a sequence of -CXXCXGXG-, which coordinate with two Zn(II) ions to form an unusual topology of two C4-type zinc fingers, C144DVC147Zn(II)C197NKC200 (Zn1) and C161PTC164Zn(II)C183PHC186 (Zn2). Studies on five DnaJ mutants with Cys in Zn2 replaced by His or Ser (C183H, C186H, C161H/C183H, C164H/183H, and C161S/C164S) reveal that substitutions of one or two Cys residues by His or Ser have little effect on the general conformation and association property of the molecule. Replacement of two Cys residues by His does not interfere with the zinc coordination. However, replacement of two Cys by Ser results in a significant decrease in the proportion of coordinated Zn(II), although the unique zinc finger topology is retained. The mutants of C183H, C186H, and C161S/C164S display full disulfide reductase activity of wild-type DnaJ, while C161H/C183H and C164H/183H exhibit severe defect in the activity. All of the mutations do not substantially affect the chaperone activity. The results indicate that the motif of -CXXC- is critical to form an active site and indispensable to the thiol-disulfide oxidoreductase activity of DnaJ. Each -CXXC- motif in Zn2 but not in Zn1 functions as an active site.